The development of an effective HIV vaccine is one of the greatest global health challenges. However despite decades of research, no vaccine has yet been able to elicit protective immunity to HIV. Recently, efforts to develop vaccines against HIV have focused on optimizing the T cell response to vaccination because of the association between T cell immunity and viral control in HIV. However, the molecular correlates of an optimal CD8 T cell response to vaccination remain unclear. This investigation will identify the molecular characteristics of optimal CD8 T cell function after vaccination, using functional genomics, cellular immunology and animal models. In Aim 1, we will generate genome-wide expression profiles of tetramer-sorted HIV-specific CD8 T cells elicited by three HIV vaccines in current clinical development. We will compare these HIV-specific CD8 profiles to antigen-specific CD8 T cells responding to vaccines against vaccinia virus (VV) - one of the most successful vaccines ever developed. We will identify patterns of genes that are unique to each type of vaccine and signatures that are common to those vaccines, such VV, that induce highly functional CD8 T cells. Candidate mechanisms that confer different functional attributes to each vaccine-induced CD8 T cell response will be identified using detailed computational analyses. This will be the first comprehensive transcriptional analysis of vaccine-induced antigen-specific CD8 T cells, and will identify new molecular correlates of highly functional CD8 T cells. Increasing data suggest that adenovirus (Ad5) vectored vaccines elicit CD8 T cells with limited functionality. In Aim 2 we will test the hypothesis that genes upregulated in CD8 T cells responding to Ad5-vectored immunogens contribute to their sub-optimal function. Based on our studies in a mouse model, and supplemented with new data from Aim 1, we will evaluate the role of up to 96 candidate regulator genes that are increased in Ad5/HIV-induced CD8 T cells using functional genomic approaches. A series of highly parallel over-expression studies and RNAi-mediated knockdown experiments will validate the role of candidate genes as inhibitory molecules in human CD8 T cells and in a mouse model of Ad5 vaccination. The proposed studies will accelerate HIV vaccine development by discovering new correlates of optimal vaccine-induced T cell immunity, and identifying the mechanisms that govern CD8 T cell function after vaccination.